CA2444955A1

CA2444955A1 - Inactivation of genes of the mep pathway

Info

Publication number: CA2444955A1
Application number: CA002444955A
Authority: CA
Inventors: Hassan Jomaa; Matthias Eberl; Boran Altincicek
Original assignee: Individual
Current assignee: Bioagency AG
Priority date: 2001-04-23
Filing date: 2002-04-13
Publication date: 2002-11-28
Also published as: JP4526763B2; US20080206267A1; DE50207641D1; ATE334209T1; WO2002095011A2; AU2002312816A1; EP1381686B1; ES2269711T3; EP1381686A2; US20110129883A1; US7875279B2; JP2004531264A; DE10119905A1; WO2002095011A3

Abstract

The invention relates to cells and organisms as well as to methods for producing said cells and organisms, according to which intermediates of the mevalonate-independent pathway for isoprenoid biosynthesis (MEP pathway) are enriched by deleting or inactivating genes. The derivatives can also be enriched by using enzyme inhibitors. The enriched intermediates may be used as substrates in enzyme activity tests. The inventive cells and organisms and t he enriched intermediates can further be used in the production of medicaments.

Description

CA 02444955 2003-10-22 f -~ i'~ ,' -. E~' ;.' _ .. .r _ 4 <'~ ' - _ Inactivation of MEP pathway genes This invention refers to cells and organisms for which intermediates of the mevalonate independent isoprenoid metabolism pathway (MEP pathway) are enriched through deletion or inactivation of genes. Furthermore, it refers to processes for producing intermediates and products derived from the MEP pathway from organisms, for which the genes according to the invention have been deleted or inactivated and genetic engineering and convention processes for producing these organisms. It also refers to the application of enzyme inhibitors for enriching MEP
pathway intermediates. It also refers to the therapeutic application of cells and organisms for which the genes or enzymes according to the invention have been deleted or inhibited and the production of medication from these cells and organisms.
The biosynthesis of isoprenoids using the classic acetate/mevalonate pathway (Beytia ED, Porter JW, Annu Rev Biochem, 1976; 45: 113-42) and an alternative, mevalonate independent biosynthesis pathway, the 2-methyl-D-erythritol pathway (MEP
pathway, synonymous with DOXP pathway) is known (Rohmer M. Nat Prod Rep, 1999 Oct;
16(5): 565-74). Both pathways lead to isopentenylpyrophosphate (IPP), the common precursor of all higher isoprenoids. While the acetate/mevalonate pathway has been known for some time and is fully understood, at present not all biosynthetic steps in the reaction of the MEP pathway are known.
In the past, various biotechnological processes have been derived, based on the application of knowledge regarding the MEP pathway:
1. Inhibitors of various enzymes through the MEP pathway are suitable as disinfectants and herbicides as the MEP pathway does not occur in humans.

2. Certain intermediates of the MEP pathway lead to a rriassive stimulation of human gamma/delta T cells. These intermediates are suitable as immunomodular medicines.

3. Through the over=expression of certain genes of the MEP pathway (e.g.
DOXP synthase, LytB), the enriching of higher isoprenes can be achieved as subsequent products of the MEP pathway.
It was previously unknown that through the deletion of a gene of the MEP
pathway or through inactivation of the corresponding enzyme, an intermediate of the MEP
pathway can be achieved.
It is known that human gamma/delta T cells are activated through one or more intermediate of the MEP pathway. This means that with the incubation of peripheral blood lymphocytes with extracts from organisms which have an MEP pathway, there is a selective proliferation and cytokine secretion of the gamma/delta T cell population (Jomaa H, Feurle J, Luhs K, Kunzmann V, Tony HP, Herderich M, Wilhelm M, FEMS Immunol Med Microbiol. 1999 Sep; 25(4): 371-8). The exact chemical composition of this activating substance of substances is still unknown.
Published data suggest that 3-formyl-1-butylpyrophosphate plays a role as a hypothetical intermediate of the MEP pathway in activating gamma/delta T cells (Belmant C, Espinosa E, Poupot R, Peyrat MA, Guiraud M, Poquet Y, Bonneville M, Fournte JJ, J. Biol. Chem. 1999 Nov 5; 274(45): 32079-84).

Consequently, it was shown that bacteria, where various genes of the MEP
pathway (e.g. DOXP reductoismerase, gepE) had been deleted, were no longer able to activate gamma/delta T cells (Altincicek B, Moll J, Campos N, Foerster G, Beck E, Hoeffler JF, Grosdemange-Billiard C, Rodriguez-Conception M, Rohmer M, Boronat A, Eberl M, Jomaa H, Jlmmunol. 2001 Mar. 15;166(6):3655-8). In order to produce these deletion mutations it is necessary to introduce genes of the mevalonate pathway using genetic engineering into the bacteria. As a result, the bacteria can then survive in the medium in the presence of mevalonate if the MEP pathway is no longer functional (fig 1 ).
Surprisingly, it was found that bacteria, whose lytB gene had been deleted (example 1 ) activated gamma/delta T cells significantly more strongly than typical bacteria (example 2, fig 2). An essential participation of the lytB gene in the MEP
pathway was displayed (example 3). A blockage of the MEP pathway at the level of the lytB
enzyme thus leads to the intermediate, which is responsible for the gamma/delta T
cells, being enriched.
Therefore, a process is available, through which the enriching of intermediates of the MEP pathway is achieved through deletion; mutation or functional inactivation of the corresponding genes. The enriching of the intermediates of the MEP pathway can also be achieved through inhibiting the enzymatic function of the corresponding polypetide.
DNA sequences, which code for a polypeptide with the amino acids represented in SEQ ID NO: 2 or for an analogue or derivative of the polypeptide according to SEQ
ID NO: 2, are particularly suitable for carrying out the process according to the process, where one or more amino acids are deleted, added or substituted by other amino acids without the enzymatic effect of the polypeptide being substantially reduced.
Furthermore, the invention is defined by claims 1-17. Further images of the invention are defined in the subordinate claims.
The following explains the invention using the enclosed figures:
Fig 1 shows the principle of enriching the intermediates of the MEP pathway through the deletion of genes of the MEP pathway. Substantial steps of the MEP pathway occurring naturally in E.coli are represented with the enzymes Dxs, Dxr, YgbP, YchB, YgbB, Gcpe, LytB. In order to be able to delete genes of the MEP
pathway, genes of the mevalonate pathway (coding for Mvk, Pmk, Mpd) are introduced through genetic engineering. Through the deletion of lytB, the intermediates are enriched which activate the gamma/delta T cells. Figure 2 shows the activation of garnma/delta T cells from the blood of healthy donors, measured as an expression of CD25, through extracts from various bacteria sources (wild type, wtDeltagcpE, wtDeltalytB) in various dilution stages. IPP serves as a control with an end concentration of 10 uM (IPP activates gamma/delta T cells substantially weaker than the intermediate according to the invention, but is suitable as a control for the test system). The wtDeltagcpE mutants were produced analogously to the wtDeltagcpB
mutants.
The genes and their genetic products (polypeptide) are listed in the sequence protocol with their primary structures and are allocated as follows:

SEQ ID NO:1 lytB-gene SEQ ID N0:2 lytB-protein The sequences come from escherichia coli.
Apart from the DNA sequences named in the sequence protocol, others are suitable which have another DNA code as a result of the degeneration of the genetic code but which code for the same polypeptide or for an analogue or derivative of the polypeptide. This also includes sequences which come from organisms other than e.coli, specifically, other bacteria, algae, plants and protozoa, and which are recognised, based on sequence comparisons or function analyses, as homologous to the sequences named in the sequence protocol.
The invention refers to cells and organisms and the production of cells and organisms, for which the genes according to the invention are functionally inactivated principally as a result of known methods. The genes do not have to be fully inactivated but instead can have their function reduced or modified. This can be achieved by the following:
~ Complete or partial deletion of the genes ~ Substitution of the gene through an artificial DNA sequence or a gene for a selection marker ~ Insertion of a gene for a selection marker ~ Mutations in the 5' and 3' area of the coding sequences (influence of promoter, enhancer, terminator sequences, ribosome conjugates) ~ Introduction of DNA constructs which code for antisens DNA
~ Application of mutagene agents, ionising radiation, UV radiation ~ Screening for spontaneous mutants The inactivation or modification of the sequences according to the invention can occur in bacteria, algae, plants and protozoa. In order to maintain stable mutants, it can be necessary to introduce the genes of the acetate/mevalonate pathway partially or in full and, if necessary, to add mevalonate or another intermediate of the MEP
pathway to the medium. Alternatively or additionally, intermediates of the MEP
pathway or derivatives or analogues to these intermediates (e.g. 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol) can be added to the medium.
Cells and organisms can also be used, which do not naturally have the MEP
pathway, if genes of the MEP pathway have been introduced through genetic engineering and bio-engineering methods. It is also possible to achieve enriching of intermediates of the MEP pathway of their derivatives by only incompletely introducing the genes of the MEP pathway. Mammal and insect cells, lower and higher fungi, slime mould and various protozoa, among others, are suitable for this.
Apart from through genetic methods, inactivation or reduction of the enzymatic activity of the polypeptides according to the invention can also be achieved through enzyme inhibitors which are added to the culture medium of the organisms or cell extracts from the organisms. The enzyme inhibitors can have synthetic or natural conjugates which reversibly or irreversibly inhibit the function of the polypeptide through competitive or allosteric interactions.

The cells and organisms according to the invention, including complete plants and parts of plants, can be reproduced and cultivated through known processes. A
co-culture with other cells or organisms, including those which do not have an pathway, is also possible. The enriched intermediates of the MEP pathway or their derivatives can be obtained through breaking down the cells or from the culture.
Various known methods are suitable for purifying the intermediates, including chromatography, electrophoreses and precipitation (e.g. as barium salts).
The enriched intermediates of the MEP pathway are suitable for various applications.
It has been found that the intermediates contain the product of the GcpE
enzyme and the substrate of the LytB enzyme. Thus, the intermediates can be used as substrates in the enzyme activity test for LytB and GcpE. In the activity test for GcpE, the reverse reaction is observed. This type of enzyme activity tests are suitable for finding GcpE
and LytB inhibitors.
The enriched intermediates of the MEP pathway are also suitable for producing medicines. The effectiveness of the substances is based on the activation of garnma/delta T cells. Depending on the area of application, the immunity can be strengthened (e.g. against tumours) or immunological tolerance against auto-antigens and allergens can be induced.
Areas of application are the treatment of immune, auto-immune diseases and allergies. For example: allergies, multiple sclerosis, rheumatoid arthritis, Hashimoto's thyroiditis; myasthenia gravis, lupus erythematosus, diabetes mellitus, primary biliary cirrhosis, active chronic hepatitis, adrenalitis/Addison's disease, polymyositis, deratomyositis, auto-immune haemolytic anaemia, myocardial and cardiac infections, sclerodema, uveitis (phacouveitis, sympathetic opthalmia), pemphigus vulgaris, pemphigoid, pernicious anaemia, auto-immune atrophic gastritis, infectious diseases of the intestines such as Crohn's disease and colitis ulcerosa, infectious diseases of the lungs such as asthmatic diseases and bronchitis.
The application is preferred for morbus Crohn, colitis ulcerosa, multiple sclerosis, asthma, chronic bronchitis, allergies.
Other applications are infections of the bone, especially osteoporosis.
The intermediates of the MEP pathway can be isolated from the organisms according to the invention or used as raw extracts for medical application. The complete organisms can also be used living or dead. The intermediates and organisms according to the invention can be used alone or in combinations with other medications. Application as adjuvant for strengthening or for modulation of an immune response is also included. Preferred methods of application are oral, inhalative and rectal application, as well as application on the skin or mucous membranes.
The following are suitable as pharmaceutical compositions: tablets, drops, capsules, pills, granules, suppositories, solutions, suspensions and emulsions, pastes, ointments, gels, creams, lotions, powders and sprays.

Tablets, drops, capsules, pills and granules can contain the active ingredients alongside the usual carriers such as (a) fillers and mixers, e.g. starch, lactose, cane sugar, glucose, mannitol and silicic acid, (b) binders, e.g.
carboxymethylcelulose, alginate, gelatine, polyvinylpyrrolidone, (c) moisturisers, e.g. glycerine, (d) explosive, e.g. agar-agar, calcium carbonate and sodium carbonate, (e) emulsifier, e.g.
paraffin and (f) re-absorption accelerator, e.g. quarternary ammonium conjugates, (g) nets, e.g.
cetylalcohol, glycerine monostearate, (h) absorbers, e.g. kaolin and bentonite and (i) lubricant, e.g. talcum, calcium and magnesium stearate and solid polyethylglycol or mixtures of the substances listed in (a) to (i). Moreover, the conjugates according to the invention can be included in other Garners such as plastics (plastic chains for local treatment), collages or bone cement.
Tablets, drops, capsules, pills and granules can be produced with the usual, if necessary opaque, casings and cases and also combined such that the active ingredients are only released, or preferably with a delay, in a specific section of the intestinal tract where embedders can be used, e.g. polymer substances and waxes.
The active ingredients can also exist in micro-capsule form in one or more of the above Garners.
Suppositories can contain the usual water soluble or insoluble carriers along with the active ingredients, e.g. polyethylglycol, fats, e.g. cocoa fat and higher ester (e.g. C 14 alcohol with C 16 fatty acid) or mixtures of same.
Ointments, pastes, creams and gels can contain the contain the usual carriers along with the active ingredients, e.g. animal and vegetable fats, waxes, paraffin, starch, tragant, cellulose derivatives; polyethylglycol, silicone, bentonite, silicic acid, talcum and zinc oxide or mixtures of same.
Powders and sprays can contain the contain the usual Garners along with the active ingredients, e.g. lactose, talcum, silicic acid, aluminium hydroxide, calcium silicate and polyamide powder or mixtures of same. In addition, sprays can contain propellants such as CFCs.
Solutions and emulsions can contain the contain the usual carriers along with the active ingredients, such as solvents, solubilisers and emulsifiers, e.g.
water, ethylalcohol, isopropylalcohol, ethylcarbonate, ethylacetate, benzylalcohol, benzylbenzoate, propylenglycol, 1,3-butylenglycol, dimethylformamide, oils, especially cotton seed oil, peanut oil, maize oil, olive oil, ricinus oil and sesame oil, glycerine, glycerine formal, tetrahydrofurylalcohol, polyethylglycols and fatty acid ester of sorbitol or mixtures of same.
Particularly beneficial is the selection of a medical application which also contains a substance which can be recognised by the immune system as a foreign object or auto-antigen.

Example 1 Construction of a lytB deletion mutant Construction of the gene exchange plasmid pK03-~lytB
In order to produce a IytB deletion mutant from E.coli, the pK03 vector was used (Link, A.J.; Philips, D.; Church, G.M.; J. Bacteriol 179, 6228-6237). In order to produce the deletion design, two sequences were amplified downstream and upstream of the lytB gene in two asymmetrical PCR stages. The primers were used in a 1:10 molar ratio (SOnM and 500 nM). Both PCR products were fused in a second PCR
amplification to form one product. The product was cloned using the pCR-TA-TOPO
cloning kit (Invitrogen) and recloned using the restriction interfaces Bam HI
and Sal I
in the pK03 vector. The following primers were used:
IytB-N-out, S'-TAGGATCCccggcctagatgactgcg-3' ItyB-N-in, 5'-CCCATCCACTAAACTTAAACAcaacaggatctgcatgttacg-3' ltyB-C-in, 5'-TGTTTAAGTTTAGTGGATGGGcgtgaagtcgattagtcat-3' ltyB-C-out, 5'-TAGTCGACagaaccacccatgatcacc-3' The restriction interfaces are underlined. Overlapping sequences, which define a 21 bp-'in frame' insertion, are printed in bold.
Construction of the synthetic mevalonate operon pSC-MVA
In order to be able to produce mutants whose individual genes of the MEP
pathway are deleted, first of all a genetically altered E. coli source was produced which was able to use mevalonate from the culture medium for the synthesis of IPP. To do this, a synthetic operon was constructed which contained the gene for the following enzyme of the mevalonate pathway from saccharomyces cerevisiae (yeast):
mevalonate kinase (ERG 12), phosphomevalonate kinase (ERGB) and diphosphomevalonate-decarboxylase (ERG19). The three genes were amplified in three asymmetrical PCR stages with genome yeast DNA as a matrix, with the primers being used in a 1:10 molar ratio (SOnM and 500 nM). Ribosome binders were included with the primers. The three PCR products were mixed so that they could hydribise with the overlapping areas and were amplified using the external primer as a fragment. The product was cloned in the pBAD vector using pBAD-TA-TOPO
cloning kits (Invitrogen) and verified using restriction and sequence analysis. The following primer set was used:
Mev-kin-Sc-for: 5'-TAGGAGGAATTAACCATGTCATTACCGTTCTTAACT-3' Mev-kin-Sc-rev: S'-TTGATCTGCCTCCTATGAAGTCCATGGTAAATT-3' Pmev-kin-Sc-for: S'-ACTTCATA GGA GGCAGATCAAATGTCAGAGTTGAGAGCCTTC-3' Pmev-kin-Sc-rev: 5'-GAGTATTATCCTCCTATTTATCAAGATAAGTTTC-3' Decarb-Sc-for: 5'-GATAAATA GGA GGTAATACTCATGACCCGTTACACAGCATCC-3' Decarb-Sc-rev: 5'-TTATTCCTTTGGTAGACCAGT-3' Overlapping sequences are printed in bold and sequences which define ribosome conjugates are in italics.
In order to check the functionality of the operon, the sensitivity to fosmidomycin from bacteria which have bee transformed with the synthetic operon was tested in the presence of mevalonate. As expected, bacteria grew, which contained fosmidomycin at a reduced rate as long as the medium contained mevalonate. Without mevalonate, the bacteria died under fosmidomycin.

Construction of the deletion mutant wt0lytB
The plasmid pK03-DeltalytB was transformed in the E. coli K-12 source DSM No.
498 (ATCC 23716), which had previously been transformed with pSC-MVA. The medium was supplemented with 100 uM mevalonate. After 1 hour incubation at 30°C, bacteria with integrated plasmid were selected through a temperature shift to 43°C. As a result of the subsequent test for sucrose resistance and chloramphenicol sensitivity, the bacteria, which had lost the vector sequences, were selected and then analysed through PCR for the desired gene type.
Example 2 Activation of gamma/delta T cells through enriched intermediates of the MEP
pathway The enriching of intermediates of the MEP pathway was detected from the ability of these intermediates to activate gamma/delta T cells. Various bacteria sources (wild type, wtDeltagcpE, wtDeltalytB) were cultivated in liquid cultures up an optical thickness of approximately 0.8. Obtaining low molecular extracts (low molecular weight, LMW) with an exclusion limit of 3kDa occurs as described ((Jomaa H, Feurle J, Luhs K, Kunzmann Y, Tony HP, Herderich M and Wilhelm M, FEMS
Immunol Med Microbiol, 25: 371 ). Lymphocytes are obtained from the peripheral blood of three healthy donors through the ficoll-density gradient centrifugation. For each test, 2 lots of 105 of the cells obtained are sown in a volume of 0.2 ml RPMI-1640-Medium (Life Technologies), which was enriched with 25 mM HEPES, 2 mM
L-glutamine, 0.025 mg/ml gentamycin, 100 U/ml human interleukin-2 (IL-2) (all from Life Technologies), and 10% human AB serum (Bavarian Red Cross). LMW
preparations were added to various solutions, IPP (sigma) was used in a final concentration of 10 uM as a positive control. The incubation was carried out at 37°C
and 5% COZ in the incubator. After 72 hours, the cells were harvested and analysed in a throughflow cytometer. The expression of the activation marker CD25 was measured on the surface of V gamma 9+ T cells using the monoclonal antibodies CD25-PE (B 1.49.9), V gamma 9-FITC (Immu360) and CD3-PCS (UCHT1 ) from the Beckman-Coulter Company. Extracts from the wild type bacteria source activated the garnma/delta T cells at a concentration of 1:500 (corresponds to approx. 2 x 10' bacteria / ml). Extracts from the DeltagcpE-deletion mutants led to a significantly reduced activation. By contrast, the activation by extracts from the DeltalytB-deletion mutants was considerable stronger than through extracts from the wild type source .
A significant gamma/delta T cell activation was also measured a concentration of 1:12500 (corresponds to approx. 8 x 105 bacteria / ml) (fig 2).
Example 3 Participation of lytB in the MEPpathway All lytB deletion mutants obtained grew strictly mevalonate dependent. In order to investigate this observation more closely, the deletion mutants wtDeltalytB
were complemented by a wild type lytB gene on a plasmid. The lytB gene was amplified with the primer eclytbfor (5'-GGATCCATGCAGATCCTGTTGGCCAAC-3') and ecltybrev (5'-AAGCTTTTAATCGACTTCACGAATATCG-3') from genomic e.coli DNA and cloned in the pCR2.1-TOPO vector. The insert was recloned through the restriction interfaces BamHI and HindIII and in the expression vector pQE30.
Bacteria from the wtDeltalytB source, which were transformed with this construct, were able to grow without mevalonate. This result confirms that lytB is an essential WO 02/09501 I PCTIEP02l04134 participant in the MEP metabolism pathway. The enriched intermediates therefore come from the MEP pathway.

Sequence protocol <i10> ,Tcneaa Fhar:na;<G GmaH
<120> I =~akt-v;z;u~g von Genen des MEN-~~Iege <13~J> 16919 <190>
<142>
<150> GG:.0119905 <151> 2:01-09-23 <1fi0> 2 <7.7C> Patentln Yer. 2,.1 <2;1.D>
<211> 951 <21?> UNA
<213> Escherich.a coli <400> 1 ai.gcagatcc tgttggc;:da C.~.C3CU~~gyG ttttg~gccg ggg=agaccg agctatcac 6~J
attyttgaaa acccgcttgc czttcacggc gcaccgztat atgtccgtcz cgasgtggtg 1.20 ca.taaccgct acgtggtcqa ~tagcctucgc gaacgi:ggag ctatci:,.l:ac tgagcagatc 180 agcr3GagCgc cgg-acggcgc gatcctgatc ~Ltctccgcac atgctg-ttc tcaggcggta 240 cgtaacgaag cgaaa3gccg tgatttgaca ~rtattcgacg c;.ac:ac~tcc gctaotyacc 3C0 asagtgcs'ta tgqaagtcg.~. cagcgcca.gc cg'ccr.;gcc aagagtctat tctca=cggt 3~a cscgccgggc accccgaa.gt ggaagggacg atgr~rr3cagt acaccaaccc tgaaggggqa 420 atgta~cy g tcgartcgcc tgacgatgtg tggaaactga cggtcaaaaa cgaagagaag 480 ctctcctt~a tgacccaaa~ cacg~~gtcg gtagatga~a cgtctgatgt gatag~cgcg S~~J
wtgcgva3ac gcttcccgaa aattg,~gge ccgcccaa~c_~ atgacatct~ ,aacgccacg 60~
act-aaccgtc agGaagcggt acgcgec=tg gcagaacags cggazqttgt gttgg'rggtc 6:0 g~-ttcga_--as act cctccaa c~Ccczaccgt ctggcgcagc tggcccagag tatgggcasa ?2Q
cgcgcgtttt tgattgacga tccgaaagal: atccagca.ag agtgggtgaa agaggttraa ?f10 tgcgtccgcg tgactgcggg cgcatcggct ccgqatattc togtgcagaa tgtggtggca 810 c=tttgcagc agctgggtgg :_gg~tcaac.~c attccgc-gg aaggccgtga agaaaatavt 900 gttttcaaag tgccgaaaga gctgcg:ctc gatattcgtq aag'_cgatta a 951 <210>
<< 11> 3..C
<?1.?> PRT
<213> 1',scheri,c,hia colf <9 C10> 2 WO 02/095011 PCTiEP02/04134 Met Gln Ile Lev ~eL, Ala ~~sn P=o .~lrg G1y the Cys AJ.a Gly val Asp l:rg Als Ile Ser I_s Val Glu Asn rlla Leu A_a _ie Tyr Gly Ala ?ro 2.0 25 3(?
Ile '1'yx ?' .1 .rg Fis GJ.u 'Jal. tTa:_ !?is Asn l~rg r~yr ~~al Val nsp Ssr Leu Arg Glu Try Gay :~la I1e Fh~: Ile Glr G1n ~le &er Glu 4a1 PLw ~0 55 c0 Asp G'_y Ala Ile Lau ?1e Phe Ser ~a tlis G_y ~:al Ser 31n rla Val 65 70 ~5 80 3rg Asn GJ.u Ala Lye 5ar Arg Asp Leu ~hr Val Phe Psp R1G Thz Cye c3 9C 95 fro Lc:u V31 Thr Lys dial 1~:is aca G1u 'Jai A-a :src Rla SEr Arg r'.r=
1.00 J.CS a.lU
Gly Glu :.-7.u Ser Il.e Leu ile Gly Eis 41a Gly Hi.s P.ro Glu t~aJ. G.l.u 115 lzo 1z5 Gly Thr i~al: Gly G1n ~_p~t Se_ Asn Fro Gl'~ G:y 31y DSe~L Tyr L:°.u Val 130 i35 . _~~"v CJ.u 3e= Fro Psp Asp Val T=p toys Leu Thr vaL yys Aen Gl~.; Clu Lys 195 X50 155 1oG
Leu Ser Phe Met Thr Cln Th= Thr Leu Se.~ val Asp Asp Thr 8e~ Asp 165 .''r '! i5 ~'al xle .'~sF Ala Leu Rrg Lys Jlrg Phe Pro Lys Ila Val Gly 'ro :arg Lys A.~,p r_sp Tle Gys Tyr A_a :'_m Thr i4sn Arg Gln Glu Ala :'al Arg P1a Leu Ala Gla Gln ?la G1a Yal Val Leu tral Yal 31,y °er Lys Aan 210 21~ ~2U
Ser Ser 1'ssn Sur Avn Frq Leu Ala G:.u heu Ala G_n Arg N~s;. 3_y hys 22: 230 235 2a0 Arg A_1.a Phe Zeu I1_e :':~;p i-~sp Ala Lys Asp Il a Gln Glu GJ.u Tr? Val 245 250 25~

Ly<_ G1u Val Lys Cy~ v'a_~ Gly ''aJ. 'i'hx rla G1.~~ ~.la Ser 3_a Prc ?~lsp I1e ~eu Val G1:1 A5n Val 'VaJ. ala Arg bet: Gln Gln ~cu ~uly Ght Gl~a 27 ~ 280 2E35 Clu ~1a Ile Pro LCU C1.L Gl.j f:rg Glu Gl~_ ~sn Ile Va? ?'ie Glu tral Fro Lys C-lu )Jc,~a ~!rg Val Aap rle Hrg G7.'.: oral ksp

Claims

claims

1. Process to enrich intermediates of the MEP pathway or derivatives of these intermediates, characterised by the fact that in selected cells and organisms a gene of the MEP pathway is deleted or inactivated or changed such that the enzymatic activity of the gene product is reduced or does not correspond to the natural activity.

2. Process according to claim 1, characterised by the fact that it involves the lytB
gene.

3. Process to enrich intermediates of the MEP pathway or derivatives of these intermediates, characterised by the fact that selected cells and organisms come into contact with enzyme inhibitors.

4. Process according to claim 3, characterised by the fact that inhibitors of the LytB
enzyme are used.

5. Process according to claims 1 to 4 characterised by the fact that for the organisms, bacteria, algae, plants and protozoa are involved.

6. Process according to claims 1 to 5 for obtaining intermediates of the MEP
pathway.

7. Process according to claims 1 to 6 for obtaining substrates for the GcpE
enzyme and the LytB enzyme.

8. Process according to claims 1 to 6 for obtaining substances which activate gamma/delta T cells.

9. Process according to claims 1 to 8 for obtaining 3-formyl-1-butypyrophosphate.

10. Intermediate, produced by a process according to claims 1 to 8.

11. Use of intermediates and derivatives of the intermediates of the MEP
pathway, obtained according to a process according to claims 1 to 9 for determining the activity of the GcpE enzyme and the LytB enzyme.

12. Use of intermediates and derivatives of the intermediates of the MEP
pathway, obtained according to one of the claims 1 to 9 for activating gamma/delta T
cells.

13. Use of intermediates and derivatives of the intermediates of the MEP
pathway, according to one of the claims 1 to 9 for producing medicines.

14. Use of living or dead cells and organisms containing intermediates or derivatives of the intermediates of the MEP pathway in an enriched concentration according to claims 1 to 9 for producing medicines.

15. Use according to claims 13 or 14 for treating human and animal diseases.

16. Use according to claim 15 for treating a disease, selected from the group comprising asthma, morbus Crohn, colitis ulcerosa, multiple sclerosis and chronic bronchitis and immune and auto-immune diseases and allergies and bone diseases and osteoporosis.

17. Use according to claim 16, characterised by the fact that a medical form of application is selected which contains additional substances which can be recognised as foreign or auto-antigen by the immune system.